Project Summary/Abstract The rising prevalence of obesity and its comorbidities is a major global health concern. The development of strategies to prevent or treat human obesity is therefore of the utmost importance. Brown adipose tissue (BAT) and its related beige fat are specialized for energy expenditure. The identification of metabolically active brown and beige fat in adult humans has positioned this tissue at the center of investigations into human energy metabolism. Considering the formidable capacity of BAT for energy expenditure and its role in fatty acid and glucose metabolism, strategies leading to increased mass or enhanced activity of BAT can potentially be utilized to combat obesity and its related metabolic disorders. Different adipose depots undergo massive remodeling in response to environmental stimuli, such as cold and diet. Prolonged cold exposure leads to recruitment of new brown adipocytes as well as a coordinated expansion and remodeling of vascular endothelium in classical BAT to enable maximal thermogenic capacity. However, the cellular origin of the cold-induced brown adipocytes, and the identity of intracellular communication pathways coordinating the adipogenesis and angiogenesis are not known. The overall goal of this proposal is to identify the role of endothelial cells in BAT expansion and remodeling in response to cold exposure and high fat diet. To address this, we used single cell RNA-sequencing (scRNA-seq) to uncover the temperature-dependent remodeling of each cell type within BAT. The preliminary data have made the novel discovery that cold exposure triggers the induction of brown adipocyte thermogenic program in endothelial cells (ECs). Additionally, the cell-type specific gene expression data allowed the identification of a Slit3-Robo4 as a potential ligand-receptor interaction mediating the crosstalk between adipocyte progenitors and ECs. The central hypotheses are that ECs contribute to cold-induced BAT expansion through de novo differentiation to thermogenic adipocytes and that Slit3 secretion from adipocyte progenitors promotes EC proliferation and angiogenesis through interaction with EC Robo4 receptor. This proposal will determine the contribution of ECs to thermogenic adipocytes pool (Aim 1) and will address the role of Slit3- Robo4 interaction in regulating adipose tissue remodeling and angiogenesis in response to high fat feeding (Aim 2). Successful completion of this proposal will change the current premise and will establish novel molecular players linking adipogenesis and angiogenesis and will have profound biomedical implications. The mentored career development award will be used to achieve a series of training objectives including expanding applicant?s knowledge in vascular biology and professional development skills that are essential for transition to an independent investigator. The training plan will build upon applicant?s expertise in adipose tissue biology and will enable the establishment of a unique cutting-edge research program in obesity and diabetes field. The team of mentors and collaborators will provide an integrative and multidisciplinary training opportunity for the applicant to receive intellectual and technical support and career development advice.